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Publication numberUS5155044 A
Publication typeGrant
Application numberUS 07/808,041
Publication dateOct 13, 1992
Filing dateDec 12, 1991
Priority dateMar 13, 1987
Fee statusPaid
Publication number07808041, 808041, US 5155044 A, US 5155044A, US-A-5155044, US5155044 A, US5155044A
InventorsStephen L. Ledis, Harold R. Crews, Timothy J. Fischer, Ted Sena
Original AssigneeCoulter Electronics, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Lysing reagent system for isolation, identification and/or analysis of leukocytes from whole blood samples
US 5155044 A
Abstract
A method and reagent system are disclosed for the rapid isolation, identification and/or analysis of leukocytes from a whole blood sample. The method and reagent system of this invention has application to any environment in which the study and/or analysis of the leukocyte fraction of whole blood requires their isolation in their native or near native state. One of the environments in which this invention can be used to advantage is in the performance of white cell differentiation on automated instrumentation designed for that purpose. In one of the preferred embodiments of this invention, the lytic reagent can contain a mixture of both formic and acetic acid, with the formic acid comprising the major component thereof and the acetic acid being present in only minor quantities, (if at all). This reagent system is used to selectively effect stromatolysis of red blood cells and create subtle modifications to the leukocyte population to enable their automated differentiation into five (5) sub-populations. The advantage and uniqueness of this reagent system is the surprising speed at which it is able to effect the foregoing objectives (generally less than 10 seconds at room temperature) and the ability to further differentiate the leukocyte population (notably, the granulocyte population). Following stromatolysis, a quenching agent is added to retard the reactivity of the lytic reagent system and, thus, inhibit any further dramatic changes to the leukocyte population. The treatment of the whole blood sample in the foregoing manner is further unique in that the leukocyte fraction of the sample has retained its characteristic immunochemical response.
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Claims(13)
What is claimed is:
1. A lytic reagent system for selective chemical treatment of whole blood, said system comprising:
an acidic aqueous solution consisting essentially of a diluent, a lytic reagent selected from the group consisting of formic acid, acetic acid and their respective mixtures; the relative concentration of the lytic reagent in said acidic aqueous solution being sufficient to effect partitioning of a whole blood sample into a lysed red cell fraction and an essentially intact leukocyte fraction in such a state as to allow differential analysis of at least five sub-populations of such leukocytes; and a clarification effective amount of saponin in the range of from about 0.05 to about 0.20 percent (w/w).
2. The lytic reagent system of claim 1, wherein the concentration of the lytic reagent in the aqueous solution is from about 0.1 to about 0.25% by volume.
3. The lytic reagent system of claim 1, wherein the aqueous solution contains up to about 0.1%, by volume, acetic acid as the lytic reagent.
4. A lytic reagent system for selective chemical treatment of whole blood, said system comprising:
an acidic aqueous solution consisting essentially of a diluent and a lytic reagent selected from the group consisting of formic acid, acetic acid and their respective mixtures;
the relative concentration of the lytic reagent in said acidic aqueous solution being sufficient to effect partitioning of a whole blood sample into a lysed red cell fraction and an essentially intact leukocyte fraction by (i) causing rapid and essentially complete hemolysis of red blood cells in such a whole blood sample and, (ii) inducing subtle changes in leukocytes in such a whole blood sample to enhance the ability of instrumentation to perform differential analysis and identification of at least five (5) sub-populations of such leukocytes, such subtle changes being effected while preserving such leukocyte sub-populations of such a whole blood sample in their essentially native physiological and/or immunochemical state; and
in addition to the acidic aqueous solution, the system includes a quench comprising a separate alkaline aqueous salt solution which is effective, upon addition to a sample lysed with the acidic aqueous solution, to rapidly retard the lytic activity of the lytic reagent, while stabilizing such a lysed sample at a pH in the range of from about 6 to about 7.5 and an osmolality in the range of from about 300 to 330 mOs.
5. The lytic reagent system of claim 4, wherein the quench comprises from about 1 to about 3% (x/v) sodium chloride and from about 0.25 to about 0.8% (w/v) sodium carbonate or bicarbonate.
6. The lytic reagent system of claim 5, wherein the quench comprises from about 1 to about 3% (w/v) sodium chloride, from about 0.25 to about 0.8% (w/v) sodium carbonate or bicarbonate, and from about 2 to about 4% (w/v) sodium sulfate.
7. A lytic reagent system for selective chemical treatment of a whole blood sample, said lytic reagent system consisting essentially of the following aqueous solutions:
(a) an acidic aqueous solution consisting essentially of a diluent and a lytic reagent selected from the group consisting of formic acid, acetic acid and their respective mixtures, said lytic reagent being present in said acidic aqueous solution at a concentration sufficient to effect partioning of a whole blood sample into a lysed red blood cell fraction and an essentially intact leukocyte fraction in such a state as to allow differential analysis of at least five sub-populations of such leukocytes; and
(b) a quench comprising an alkaline aqueous solution which, upon addition to a whole blood sample lysed with the acidic aqueous solution stabilizes such a lysed sample at a pH in the range of from about 6 to about 7.5 and at an osmolality in the range of from about 300 to 330 mOs.
8. The lytic reagent system of claim 7, wherein the concentration of the lytic reagent in the aqueous solution is from about 0.05 to about 0.5% by volume.
9. The lytic reagent system of claim 8, wherein the aqueous solution contains up to about 0.1%, by volume, acetic acid, as the lytic reagent.
10. The lytic reagent system of claim 7, wherein the concentration of the lytic reagent in the aqueous solution is from about 0.1 to about 0.25% by volume.
11. The lytic reagent system of claim 7, wherein, in addition to the lytic reagent, the aqueous solution contains a clarification effective amount of saponin in the range of from about 0.05 to about 0.20% (w/w).
12. The lytic reagent system of claim 7, wherein the quench comprises from about 1 to about 3% (w/v) sodium chloride and from about 0.25 to about 0.8% (w/v) sodium carbonate or bicarbonate.
13. The lytic reagent system of claim 7, wherein the quench comprises from about 1 to about 3% (w/v) sodium chloride, from about 0.25 to about 0.8% (w/v) sodium carbonate or bicarbonate, and from about 2 to about 4% (w/v) sodium sulfate.
Description

This application is a continuation of application Ser. No. 07/317,147, filed Feb. 28, 1989, now abandoned, which is a continuation of application Ser. No. 07/025,303, filed Mar. 13, 1987, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to compositions of matter and methods employing one or more of these compositions. More specifically, this invention concerns a novel lytic reagent system for rapidly effecting hemolysis of the erythrocyte fraction of whole blood and thereby permitting the isolation of the leukocyte fraction in its native or near native state. The leukocyte fraction may then be subjected to further study or analysis in a variety of environments. One of the preferred uses of this lytic reagent system is the pretreatment of the whole blood sample to effect rapid and essentially complete hemolysis of the erythrocyte fraction. Such pretreatment also results in subtle modification to the leukocyte fraction, thus facilitating its further differentiation into at least five (5) distinct sub-populations.

2. Description of the Prior Art

The separation of complex biological fluids (i.e. whole blood) into its various constituents preliminary to study and analysis of its component parts, is generally desirable and often an essential requirement of many established analytical protocols and/or instrumentation utilized in such studies and analysis. Where such study or analysis of the fluid fraction of the sample is of primary interest, the cellular fraction is separated from the sample without regard for maintaining the cell viability or membrane integrity. Conversely, where the cellular fraction is itself of primary interest to the researcher or clinician, the partitioning of the whole blood sample into its various cellular components requires that the sample treatment/processing techniques be adjusted accordingly. The traditional methods for separation of whole blood samples into its various cellular components is by centrifugation. While this process is effective, it is labor intensive, relatively inefficient and requires physical manipulation of the cellular fraction of the sample.

Where such separation/partitioning of the cellular fraction of the sample is attempted with chemical agents, the results have been less than totally satisfactory for a variety of reasons. More specifically, the vitality and viability of a cell population in vivo or in vitro is dependent upon maintaining a precise physiological environment that is consistent with the preservation of physical cell structure and chemical balance within the cell. This balance is controlled by the permeability and transport characteristics of the cell membrane. Alteration in the physiological environment will in turn evoke a response or change in the cell membrane. The membrane response is "defensive" in nature; that is, the physiological response of the membrane is calculated to maintain the chemical balance within the cell and, thus, its continued and uninterrupted vitality.

It is fully appreciated that such alteration in the ideal physiological environment of the cell can be tolerated only within limits; and, that when such limits are exceeded, permanent injury to the cell can occur. As is further appreciated in the art, such changes in environment (i.e. tonicity of the diluting medium--even with distilled water) can effect hemolysis of the cells.

The degree of tolerance of various cell populations in whole blood to changes in their physiological environment has been extensively studied and documented. The effects of alteration in various aspects of the physiological environment of the cellular fraction of whole blood are both subtle and dramatic and can be effected through dietary metabolites and/or foreign substances. These studies include monitoring the reaction of cellular preparations to different drugs, Da Costa, A. J. et al, Transfusion, (1973), 13, 305; to dietary imbalance, Kobayashi, T. et al, Journal of Biochemistry (1983) 93, 675; and to changes in pH, Rother, U. et al, Z. Immunologie Forschungsgemeinschaft (1978) 155, 118, and Schettini, F. et al, Acta Paediat. Scand. (1971), 60, 17.

In each of the articles noted above, a drug, food metabolite or change in pH resulted in significant alteration in the physiological environment of the blood to the degree where hemolysis of the red blood cells was effected.

More specifically, the above referenced Rother article reports serum activation by acidification (pH 6.4) with hydrochloric acid which lysed unsensitized erythrocytes in the presence of EDTA. The article compares the effect of such acidification with the "deviated lysis" activity observed following serum activation with insulin. An independent and unrelated study by Schettini and his co-workers concluded that red blood cells from infants and young children were more sensitive to acid hemolysis than red blood cells from older individuals.

As of the present, no chemical treatment is presently available to rapidly and effectively partition a whole blood sample into viable cellular fractions. Where one or more chemical treatments of the whole blood sample is used (as in the preparation or pretreatment of whole blood for the performance of white blood cell differentials), the focus of such treatment has been to alter the sample to permit the differentiation of its cellular components from one another based upon isolation/analysis of (i) the cellular debris (i.e. nuclei) remaining subsequent to such treatment; (ii) the fixation of the cells by such chemical treatments; or (iii) the relatively severe modification of such cells which eventually results in their ultimate disintegration. Such relatively harsh and disruptive chemical treatments of whole blood samples have, however, been successfully applied where combined with relatively sophisticated instrumentation. More specifically, the ability to alter the physiological environment of a cellular population in vitro has been used to advantage in the measurement of certain cellular parameters and to quantitate the individual populations. The distinctive reaction of each individual cell population to a change in its physiological environment has particular advantages in the identification of the individual leukocyte sub-populations of cells of whole blood. The leukocyte population of blood has been classified previously into two major fractions: the lymphoid and the myeloid fraction. The lymphoid fraction consists of lymphocytes (B and T cells). The myeloid fraction consists of monocytes and granulocytes (neutrophils, basophils and eosinophils). One accepted technique for modification of the physiological environment of the cellular population of whole blood has been through the addition of certain so-called "lytic reagents" to a blood sample. The development of certain lytic reagents and lytic reagent systems has provided the clinician with the ability to effectively isolate the white cell population (hereinafter "leukocytes") from the red cell population of whole blood. The relative concentration of leukocytes within the blood sample and the gross morphological appearance of certain classes of these cells can be clinically significant.

This ability to further differentiate the leukocyte population, thus, provides an invaluable diagnostic tool in the study and treatment of various diseases. As is further appreciated, the larger the number of sub-populations of leukocytes which are identifiable, the more accurate and reliable the identification of any one such sub-population.

A number of references have appeared in the recent patent literature which disclose various reagent systems and techniques for enhancing the ability of automated instrumentation to conduct white blood cell differentials. The following references are representative of the pertinent patent literature in this field: U.S. Pat. Nos. 3,874,852; 4,286,963; 4,346,018; 4,485,175; 4,520,274; 4,529,704; and U.S. applications Ser. No. 615,961 (corresponding International Application PCT/US85/00954, published Dec. 19, 1985); and, Ser. No. 615,966 (corresponding International Application PCT/US85/00868, published Dec. 19, 1985), all of which are hereby incorporated by reference in their entirety.

U.S. Pat. No. 3,874,852 (to Hamill) describes a reagent system and method useful in the performance of leukocyte and hemoglobin determinations of whole blood. This reagent system comprises an essentially ferrocyanide free aqueous solution of quaternary ammonium salt and cyanide ions. This reagent system is effective to stromatolyze both red blood cells and platelet cells in whole blood and in the conversion of the free hemoglobin to a chromogen. This system is reported efficacious for leukocyte and hemoglobin determinations with diagnostic accuracy. The leukocyte population profile available with this system is, however, limited to total white cell count, without further differentiation of this cellular fraction into its discrete sub-populations.

U.S. Pat. No. 4,286,963 (to Ledis, et al) describes a lytic diluent and method for achieving rapid lysis of red blood cells in whole blood. This diluent enhances the ability of automated instrumentation to perform differential determinations of lymphoid and myeloid sub-populations of leukocytes and the quantitative determination of hemoglobin. The lytic diluent described by Ledis is composed of a mixture of at least one quaternary ammonium salt and an aryl substituted short chain alkanol in buffered aqueous medium (pH 3.5 to 5.0). The lytic diluent of this Ledis patent is, however, limited in its ability to effect differentiation of the leukocyte population into the two (2) principle sub-populations; namely, the lymphoid and myeloid fractions.

U.S. Pat. No. 4,346,018 (to Carter, et al) describes a multipurpose blood diluent and a method for utilizing this diluent in combination with a weak lysing reagent system for the performance of hemoglobin determination and the differentiation of lymphocytes into the lymphoid and myeloid sub-populations. This diluent comprises, among other constituents, N-(2-acetamido)iminodiacetic acid (ADA) as a blood stabilizing agent. The lysing agent comprises an aqueous solution of at least one quaternary ammonium salt. The diluent/lytic reagent of this Carter patent is, however, limited in its ability to effect differentiation of the leukocyte population into the two (2) principle sub-populations; namely, the lymphoid and the myeloid fractions. In addition, ADA has been found to help stabilize the size distribution, cellular shape, and most importantly, the high degree of cellular dispersion of erythrocytes and platelets to an extent not previously observed with other compounds.

U.S. Pat. No. 4,485,175 (to Ledis, et al) describes a reagent system and method for performance of differential determinations of leukocytes into three (3) sub-populations utilizing automated cell counting equipment. This reagent system comprises a blood diluent and a lysing agent. The lysing agent (comprising an aqueous mixture of quaternary ammonium salts), when added to the diluted blood sample under mild conditions of concentration and at a relatively slow rate, causes unexpected volume modifications to the various leukocyte sub-populations. The discovery which permitted the attainment of the degree of differentiation of the leukocyte population by Ledis, et al is based upon the observation of the relative greater sensitivity of the granulocyte sub-population to lytic agents. By controlling the rate of exposure of the lymphocyte population to lytic agents, the granulocyte sub-population is better preserved. The reagent system of this Ledis et al patent is, however, limited in its ability to effect differentiation of the leukocyte population into three (3) sub-populations; namely, lymphocytes, monocytes and granulocytes.

While all of the above lytic agents and reagent systems facilitate the differentiation of the leukocyte fraction of a blood sample (to a greater or lesser degree), each suffers from a common deficiency; namely, the inability to effect such differentiation without adversely altering the chemical balance of the cells which are subjected to such treatment. Where such alteration in the chemical balance is induced, the effect on the cellular population can range from relatively minor changes (i.e. swelling) to lysis. Dramatic chemical changes in the physiological environment of the leukocyte population also alters the immunochemical response of the leukocyte surface markers. The treatment of leukocytes with such traditional lytic agent system is, thus, inherently incompatible with further immunochemical study of these leukocytes. This limitation has thus, up to now, prevented the use of lytic reagents, alone or in combination with other means, for further refinement in the diagnostic process of various disease states, based upon the differences in the immunochemical response of the respective surface markers of each such cell population.

OBJECTS OF THE INVENTION

Accordingly, it is the object of this invention to remedy the above as well as related deficiencies in the prior art.

More specifically, it is the principle object of this invention to provide a chemical treatment, or pretreatment, of a complex biological fluid sample, such as whole blood, which facilitates the subsequent isolation, identification and/or analysis of one or more cellular populations that are present in the fluid sample.

It is another object of this invention to provide a chemical treatment, or pretreatment, of a whole blood sample which facilitates the partitioning of the sample and thereby the subsequent isolation, identification and/or analysis of the leukocyte fraction based upon the physical, physiological and/or immunochemical properties of such fraction in its native or near native state.

It is yet another object of this invention to provide a chemical treatment, or pretreatment of a whole blood sample which is selective for only one of the cellular constituents of sample.

It is still yet another object of this invention to provide a reagent system which can rapidly and efficiently partition a whole blood sample into an essentially intact leukocyte fraction and a lysed erythrocyte fraction.

It is an additional object of this invention to provide a lytic reagent system for use in differential determination of leukocyte sub-populations of whole blood.

It is another additional object of this invention to provide a novel lytic reagent system which includes both a lytic reagent and a companion quench for use in differential determination of leukocyte sub-populations in whole blood.

It is yet another additional object of this invention to provide a novel lytic reagent system which is effective for use in differential determination of leukocyte sub-populations in whole blood by (a) measurement of physical and/or optical properties of such sub-populations, and/or (b) observation of the immunochemical response or interaction of such sub-populations with immunoreagents (i.e. antiserum) specific for one or more surface markers on each such cell sub-populations.

It is a further object of this invention to provide a method for performance of differential determination of leukocyte sub-populations by both (a) measurement of their physical and/or optical properties, and (b) immunochemical response of such sub-populations to antiserum specific for one or more surface markers on each said sub-populations.

SUMMARY OF THE INVENTION

The above and related objects are achieved by providing a chemical reagent system which is selective in its interactions with the various cellular components of a complex biological fluid sample. This reagent system will, in the various environments contemplated for its use, provide at least one reagent component to effectively alter, on a selective basis, one or more cellular constituents of the complex biological fluid sample to the degree necessary to permit the subsequent isolation, identification and/or analysis of the cellular constituents of interest. This reagent system further contemplates the ability to modulate the chemical treatment of the cellular constituents of the sample by providing a separate reagent which is designed to quench or retard the action of the cell altering reagent on the cellular populations of the sample.

The principles and concepts of this invention have been successfully applied to the treatment of whole blood samples preliminary to white blood cell differential analysis. Where this chemical reagent system is used for performance of such differential analysis, it is hereinafter referred to as "lytic reagent system"; and, the separate reagent used to retard the activity of the lytic reagent is referred to by the same designation as before ("quench").

The lytic reagent system and method of this invention, thus, has as its broadly stated objectives, the selective hemolysis of the erythrocyte population of a whole blood sample, while facilitating the subsequent isolation, identification and/or analysis of one or more of the leukocyte sub-populations of the same sample, based upon one or more physical, physiological and/or immunochemical characteristics which are indicative of the sub-population of interest. In this preferred embodiment of the concepts of this invention, the novel lytic reagent system comprises an aqueous solution containing a differentiation effective amount of lytic reagent selected from the group consisting of formic acid, acetic acid and their respective mixtures. In these mixtures the formic acid will preferably comprise the major functional component with the acetic acid being present in only minor amounts. The phrase "differentiation effective amount" is used throughout this disclosure as indicative of a concentration of lytic reagent which is not only effective for lysing red blood cells, but also effects subtle changes in the leukocyte cell fraction to facilitate the subsequent isolation, identification and/or analysis of this leukocyte cellular fraction; including the ability of instrumentation to perform differential analysis and identification of at least five ( 5) sub-populations of leukocytes. The preferred range of concentration of formic acid in the lytic reagent is in the range of from about 0.10 to about 0.25% (v/v). The concentration of such preferred lytic reagent which has been determined as satisfying the foregoing criteria is from about 0.009 to about 0.020 milliliters of formic acid per milliliter of whole blood. This subtle modification of the leukocyte fraction by the lytic reagent is achieved while preserving the immunochemical response of the surface markers of each of the five (5) cell sub-populations of leukocytes.

As stated previously, it is the primary objective in this treatment of the whole blood sample with the lytic reagent, that such reagent effectively accomplishes stromatolysis of the erythrocyte cell fraction while preserving the leukocyte fraction in its essentially native state.

In one of the preferred embodiments of this invention, the reagent system can contain saponin in addition to the lytic reagent. The term "saponin" is intended as referring to commerical grade quillaja saponin powder. The addition of saponin to the reagent system is optional and generally only appropriate where the clinician is monitoring certain parameters of the leukocyte sub-populations other than by photooptical or immunochemical techniques. The addition of appropriate quantities of saponin to the lytic reagent system is effective in its ability to reduce the size of red cell fragments so as to prevent their interference in determination of certain leukocyte parameters by measurement of electrical opacity and/or Coulter volume utilizing the techniques described in Coulter U.S. Pat. Nos. 2,656,508 and 3,502,974 (which are hereby incorporated by reference in their entirety). The preferred range of concentration of saponin which has been determined as effective for reducing red cell fragments is from about 0.006 to about 0.012 grams per milliliter of whole blood. In brief, the technique involves the measurement of cell volume utilizing radio frequency current (RF) and DC field excitation. By generating a particle sensing field with both a low frequency or direct current (DC) and radio frequency (RF) current excitations, two or more interrelated output signals can be derived from passage of a single particle (i.e. leukocyte) through an electric field. The value derived from this output signal is termed "relative opacity", which is unique for each sub-population of leukocyte. The addition of saponin to the lytic reagent reduces the size of the red blood fragments to a point where they will not interfere with or themselves cause, the derivation of an output signal indicative of a species of leukocyte.

Where cell differentiation is based upon light scatter measurements, (utilizing the techniques described in Fulwyler U.S. Pat. No. 3,989,381 and/or Auer, et al U.S. Pat. No. 4,038,556--which are hereby incorporated by reference in their entirety), the red cell fragments do not interfere or adversely effect the photometric differentiation of the various sub-populations of leukocytes. Thus, the addition of saponin to the lytic reagent system is unnecessary where such differentiation is based upon photometric analysis.

The amount of time of exposure of the blood sample to the lytic reagent system is critical to the differentiation method of this invention. This exposure period, as illustrated in the Examples disclosed hereinafter, should not exceed ten (10) seconds, and most preferably, requires only about six (6) seconds or less. Both of these exposure times are specified for room temperature (˜18°-28° C.). In each instance, the action of the lytic reagent is quenched by simple addition of appropriate concentrations of salts to the sample to return the cells to their native physiological environment. The quench effectively retards further activity of the lytic reagent upon the leukocytes without need for the addition of fixatives. The quench for the lytic reagent is an essential complement to the lytic reagent system where subsequent analysis of the leukocyte fraction requires retarding the activity of the lytic reagent. The leukocytes are stabilized by this quench by controlling the pH within a fairly narrow range (pH ˜6.00 to 7.25) and osmolality (˜300 to 330 milliosmoles). The quench can also be formulated to match the conductivity of a chosen "sheath" fluid which is utilized in a focused flow aperture analysis system. The composition and volume of the quench is adjusted to provide optimal separation of the five (5) major leukocyte subclasses when analyzed in accordance with the techniques (RF frequency current in combination with DC field excitation) described in Coulter, et al U.S. Pat. No. 3,502,974 (previously incorporated by reference). The leukocyte fraction of the sample, treated in the above manner, can be readily differentiated to at least five (5) sub-populations by a hematology analyzer capable of multiple parameter particle (cell) measurements; and, by immunochemical interaction with antiserum (i.e. antibodies, binding proteins, etc.) that are specific for one or more surface markers on the cells of each such cell sub-population.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2 and 3 are scattergrams representing the white cell differential analysis of the blood sample of Example II; the X-axis of each such Fig. being different from one another.

FIG. 4 is a scattergram representing the white cell differential analysis of the blood sample of Example I.

FIG. 5 is a scattergram representing the white cell differential analysis of the blood sample of Example V.

FIG. 6 is a scattergram representing the white cell differential analysis of the blood sample of Example VI.

DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS

The lytic reagent system of this invention comprises an aqueous solution containing surprisingly low concentrations of the lytic reagent (preferably less than 1.0% by volume). The lytic reagent is preferably formic acid, acetic acid or mixtures of formic and acetic acid in which formic acid is the predominant functional component. This aqueous solution is prepared by simple addition of the lytic reagent to deionized water. The amount of lytic reagent added to this diluent is sufficient to prepare a solution containing from about 0.05 to about 0.5% (v/v) lytic reagent. In the preferred embodiments of this invention, the concentration of lytic reagent will range from about 0.1 to about 0.25% (v/v). The concentration of acetic acid used may vary from about 0 to about 0.1% by volume.

Where the lytic reagent comprises a mixture containing both formic and acetic acid, the acetic acid is preferably only present as a partial replacement for a definitive amount of formic acid and then only at a concentration in the range of from about 0.05 to 0.10% (v/v).

The lytic reagent system also optionally can contain very small amounts of saponin (preferably at least about 0.05 up to about 0.2 weight percent) in addition to the lytic reagent. As is fully appreciate, the effectiveness of saponin, as a lytic reagent, is highly concentration dependent. If saponin is used at low concentrations, it is generally ineffective for this purpose. Were saponin to be used as a lytic reagent, it would have to be present in concentrations of at least 2.0 weight percent, or more, to effect essentially complete stromatolysis of the red blood cells. Unfortunately, the saponin, where present at a lytic effective concentration, can also cause lysis of white blood cells. Thus, any changes induced in the white blood cell population utilizing saponin as the lytic agent, and their subsequent differentiation by automated instrumentation must be based upon whatever discernible and distinctive characteristic of their respective nuclei. Its presence in the lytic reagent system of this invention is, however, desirable where the clinician is concerned with so-called "ghosts" (intact red cell membranes) interfering with differential measurements based upon distinctions in physical and/or electrical properties. As noted previously, saponin would not be a necessary addition to the lytic reagent system if the differential measurements where made by purely photooptical instrumentation and/or immunochemical analysis.

The lytic reagent system can also contain other traditional additives, to the extent their presence is not otherwise incompatible with the primary functional components of the system (i.e. anti-microbial preservatives, such as formaldehyde or sorbic acid).

The lytic reagent system of this invention can be combined with a whole blood sample by simple manual or automated addition, the lytic reagent and sample allowed to briefly interact, and the action of the lytic reagent substantially retarded by addition of a suitable quench. The quench, to be effective in this environment, must, thus, be capable of retarding the lytic activity of the lytic reagent immediately upon its addition to the aqueous mixture contained in the blood sample and the lytic reagent. The precise formulation of the quench can vary, depending upon the composition of the lytic reagent system and the sheath fluid used in a focused flow aperture analysis system. The quench is typically an aqueous solution containing soluble salts which are both effective to substantially retard and/or substantially neutralize the lytic activity of the lytic reagent and restore the ionic balance to the sample. This restoration of the ionic balance will extend the longevity of the surviving cells and permit subsequent analysis on equipment which requires that the sample be electrically conductive (contain electrolytes), as for example with a Coulter Counter® whole blood analyzer.

A quench which is suitable for use in conjunction with a lytic reagent composition of this invention can, and usually will, contain any combination of at least two of the following four ingredients: sodium chloride, sodium sulfate, sodium bicarbonate, sodium carbonate; and, in addition, sodium azide as a preservative. The effectiveness of the quench upon the lytic reagent in the context of this invention is determined by its ability to rapidly reduce the lytic activity of the formic acid, acetic acid and the mixtures of formic and acetic acid. As noted above, the method and equipment utilized in the differentiation of the leukocyte sub-population can also place certain requirements (i.e. conductivity, pH, etc.) upon the precise formulation of the quench. More specifically, when such differentiation is performed in a focused flow aperture analysis system, the composition and volume of quench can be critical to optimal separation (differentiation) of the five (5) leukocyte subclasses from one another. In this type of analysis system, the ionic balance of the quench must also be adjusted to obtain a satisfactory conductivity match of the lysed blood sample to the sheath fluid. In the preferred quench formulation, the major ionic species and their relative ratio in the lysed-quenched blood sample should be essentially the same as the major ionic species and their relative ratio in the sheath fluid. The relative concentration of the functional components of the quench, which is to be used in conjunction with the lytic reagent system of this invention, will range from about 1 to about 3% (w/v) sodium chloride, about 0.25 to about 0.8% (w/v) sodium carbonate or bicarbonate, and about 2 to about 4% (w/v) sodium sulfate. The precise relative quantities of ingredients of the optimum quench are generally determined empirically; the objectives of such adjustment being to attain the pH of the lysed blood sample within the range of from about 6.0 to about 7.5, and a final osmolality of the stabilized lysed blood sample in the range of from about 300 to about 330 mOs. It has been previously observed, in a focused flow aperture system, that optimal clustering of the leukocyte subclasses is achieved by adjustment in the osmolality of the final blood sample to about 310 mOs. The essentially complete neutralization of the acidity of the lysed sample with an alkaline quench can be critical to a focused flow aperture analysis system. Components of the sample (i.e. fibrin and platelets) are pH sensitive and can form aggregates under acidic conditions which can potentially interfere with differential analysis (i.e. noise) or physically obstruct the aperture of a focused flow aperture system.

It is not contemplated, nor intended, that the quench also necessarily inhibit or neutralize the saponin (when present). The reasons for this are quite simple, in that the saponin, at the concentrations contemplated (˜0.2 weight percent), is not completely effective as a lytic reagent. If the quench were to inhibit the saponin activity upon its addition to the sample, adequate clarification of the sample (destruction of intact red blood cell membranes) would not take place. Thus, it is anticipated that the activity of the saponin continue for about 5 to 15 seconds subsequent to quenching of the activity of the lytic reagent. It may, under some circumstances, be appropriate to provide an independent quenching agent for the saponin; however, at the concentrations presently contemplated, (less than 0.1%) none would appear necessary to achieve or further the objectives of this invention.

In the preferred embodiments of this invention, the duration of effective contact of the lytic reagent and the blood sample (from the time the two are combined, to the time when the quench is added), must be less than ten (10) seconds, and most preferably six seconds or less. The interval of reactive contact of the lytic reagent and blood sample as specified above, presumes such reactive contact occurs at room temperature (˜18°-28° C.). Of course, if the temperature is in excess of this level, the period of reactive contact would be somewhat less and inversely, if the temperature is lower than this level, the period of reactive contact would be somewhat longer. It is both critical and essential to the successful performance of the differential method of this invention, that the kinetics of the interaction of the lytic reagent upon both the sacrificial cell population (red blood cells) and the desirable cell fraction (leukocytes) be controlled carefully and precisely. The mechanism by which the lytic reagent reacts with both cell fractions is not known with precision, only the manifest effect of such interaction. It is, thus, beyond the scope of this discussion to speculate how these improvements in differential analysis are attained and, thus, no attempt is made herein to explain or later claim such mechanism.

By limiting the exposure of both these cell fractions to the lytic reagent, stromatolysis of the erythrocytes is effectively and efficiently accomplished, while additional subtle changes are induced in the leukocytes by the quench to enable their effective differentiation. Both of these events occur essentially concurrently, while preserving the native immunochemical reactivity of the differentiated leukocyte fraction.

As noted above, the duration of contact of the lytic reagent with the blood is sufficient to effect a selective destructive response of the sacrificial cell fraction, while at the same time effecting a differential response in the leukocyte fraction; such changes unexpectedly permitting the physical differentiation of at least five (5) cell sub-populations of leukocytes from one another. The lytic reagent, in sharp contrast to the more traditional types of lyse (i.e. saponin, quaternary ammonium salts), does not detrimentally alter the native immunochemical response of the surface markers of each of the cells within the leukocyte sub-populations. This quality is believed to be unique to the lytic reagents of this invention.

Blood samples which have been exposed to the above lytic reagent system can be subjected to differential measurement on instrumentation designed for this purpose. Such differentials can be performed on a device utilizing technology of the type described in U.S. Pat. Nos. 3,549,994 (which is hereby incorporated by reference in its entirety), 3,502,974 and 3,989,381. Features of the inventions described in the above referenced patents can be embodied into commercially practical instrumentation. In brief, the blood sample is initially treated by mixing with the lytic reagent system, utilizing automated pipetting equipment. The lytic action of the lytic reagent on the sacrificial cell population (red blood cells), must be both rapid and effective. A quench then is added by similar pipetting means to substantially retard the activity of the lytic reagent upon the surviving cell fraction (leukocyte). The sample containing the leukocyte fraction then is subjected to counting of each of the individual cell sub-populations, and/or histograms and/or scattergrams generated from the data collected in this fashion.

The principle involved in performance of Coulter volume measurements are well known to those skilled in the art as the Coulter Principle. In brief, the operation of instrumentation utilizing the Coulter Principle involves the measurement in the impedance caused by the passage of individual cells through a sensor designed to detect a voltage drop caused by the presence of the cell. The instrumentation utilizing this principle comprises two fluid vessels or chambers, each containing a conductive electrolyte solution. At least two electrodes having opposite polarity are immersed in the electrolyte solution, with each fluid compartment having one of the electrodes disposed therein. A sample of the electrolyte solution, having the blood cells suspended therein, is passed through a constricted fluid path, or orifice, interposed between the two fluid compartments. Although the constricted pathway can take different forms, in each device such path defines a sensing zone wherein the presence or absence of a particle gives rise to a detectable change in electrical characteristics of the path. For example, relatively poorly conductive blood cells passing through this path, displace a volume of electrolyte solution equal to the cell volume, causing a voltage drop by increasing the path impedance. The resistance pulses defined by the drops in voltage are used for particle counting and particle volume determination. The Coulter principle is more fully described in U.S. Pat. No. 2,656,508. This technique for sensing and identifying specific cell populations can be enhanced by a combination of Coulter principle measurements with radio frequency excitation of the cells within the sensing zone. In brief, this radio frequency enhancement operates upon the principle that a particle moving through the sensing zone of a hematology analyzer will cause a phase shift in radio frequency (RF) energy within the sensing zone. This shift in phase can be correlated with physical and compositional characteristics of a cell population. This technique for RF differentiation of cells is more fully described in U.S. Pat. No. 3,502,974. White cell differentiation also can be achieved utilizing optical measurement principles of flow cytometry as described in U.S. Pat. No. 3,380,584 (which is hereby incorporated by reference in its entirety).

The lytic reagent system of this invention is effective to induce subtle changes in the leukocyte cell fraction to enhance the differentiation of five (5) distinct sub-population of leukocytes on automated cell counting equipment, see for example, U.S. Pat. No. 4,412,004 (to Ornstein, et al--which is hereby incorporated by reference in its entirety). These five (5) distinct sub-populations, as noted previously, include: lymphocytes, monocytes and three species of granulocytes (eosinophils, basophils and neutrophils). Each of these sub-populations of leukocytes have distinctive surface markers. In certain disease states, the surface markers on one or more of these sub-populations will provide a unique immunochemical response and, thus, make diagnosis or confirmation of disease possible at an early stage of its development. The detection of these distinctive surface markers on one or more of these sub-populations of leukocytes will, of course, be dependent upon the ability to effectively physically isolate these cells having characteristic disease state surface markers from the non-effected cells; and, the relative concentration of the effected cells within the sample being analyzed.

The initial lytic conditions contemplated by this invention permit an extension in the longevity of the cell populations which survive and which are to be differentiated from one another. For the most part, it is anticipated that cell longevity of at least seventy-two (72) hours would be appropriate for certain types of immunochemical analysis. In certain applications of this differential method, it may be both necessary and appropriate to maintain one or more of these sub-populations in vitro for several hours, or possibly even several days. In order to achieve such extended stability, it is advisable to physically separate the leukocytes from the fluid fraction containing the lytic reagent/quench mixture and, thereafter, resuspended such cells in a physiological medium.

The following examples are provided as illustrative of the unique advantages of the lytic reagent system of this invention. The equipment and techniques utilized in the preparation and evaluation of this lytic reagent system are standard or as hereinbefore described. Parts and percentages appearing in such Examples are by weight unless otherwise stipulated.

EXAMPLE I

The lytic reagent system of this invention was prepared from reagent grade chemicals. A 0.12% (v/v) formic acid solution was initially prepared by combining 1.3 ml of 90% formic acid and 998 ml deionized water. A 50 uL whole blood sample (K3 EDTA) and 800 microliters of 0.12% formic acid solution are gently mixed by swirling the two together for five (5) seconds at room temperature (˜20° C.). The lytic action of the formic acid arrested after about 5 seconds, by the addition of 400 ul of a quench solution containing 0.55% sodium bicarbonate, 3.0% sodium chloride and 0.01% sodium azide. The sample was adequately quenched and ready for differential analysis by flow cytometry techniques within about 5 to 10 seconds subsequent to the addition of the quench. The equipment used in such differential analysis was equipped with a helium/neon laser and silicon diode detectors for measurement of light scatter. The leukocytes are observed and their individual parameters determined by optical parameter analysis, i.e. measurement of light extinction (zero angle scatter), and any one of several angular ranges of light scatter. The scattergram of the sample generated in the above manner is illustrated as FIG. 4. Five (5) distinct sub-populations of leukocytes are identified and quantified in this scattergram.

EXAMPLE II

The procedures of Example I are repeated, except for the addition of 0.05% (w/v) saponin powder to the aqueous solution containing 0.12% formic acid. The whole blood sample (50 microliters) is then combined (as previously described) with 600 microliters lytic reagent. The lytic reagent is quenched after 6 seconds by addition of 265 microliters of an aqueous solution containing 0.60% sodium carbonate, and 3.00% sodium chloride.

The addition of saponin to the lytic composition effectively eliminates interference of the red cell debris from Coulter volume measurements. The effectiveness of the saponin is highly temperature dependent. The essentially total elimination of interference from red cell debris requires an additional 10 seconds (at room temperature ˜18° to 28° C.) subsequent to completion of lysing the red cell fraction of the sample. In the event the sample is maintained at a lower temperature (below 18° C.), a somewhat longer period will be required to effectively eliminate interference from the red cell debris by the saponin.

The sample was ready for differential analysis within about 10 to 20 seconds subsequent to the addition of the quench. The sample was subjected to photo-optical measurement as described in Example I. Leukocytes in the sample were also observed by measurement of DC and RF volumes using an ISOTON® II sheath fluid and the resultant scattergram is illustrated in FIG. 1. Four distinct sub-populations of leukocytes were identified and quantified in the simultaneously obtained light scatter vs. DC scattergram of FIG. 2. A fifth sub-population of leukocyte (basophils) is isolated by generation of a "gated" secondary scattergram. This basophil population is depicted in the scattergram illustrated in FIG. 3.

EXAMPLE III

The procedures of Example II are repeated, utilizing the same formic acid/saponin reagent composition. The lytic activity of the formic acid is arrested with a quench containing 3.13% (w/v) sodium sulfate (anhydrous), 1.45% (w/v) sodium chloride and 0.60% (w/v) sodium carbonate (anhydrous). The sample was analyzed by electro-optical techniques in the manner described in Example II and the scattergram results were similar to that of FIGS. 1, 2 and 3. The sheath fluid was, however, changed to ISOTON® III diluent.

EXAMPLE IV

The procedures of Example III were repeated, except for use of a concentrated lysed-quenched blood sample for more rapid data acquisition. The lytic reagent comprises 0.15% (v/v) formic acid and contains 0.10% (w/v) saponin powder. The lytic activity of the formic acid was arrested by a quench containing 2.67% (w/v) sodium sulfate (anhydrous), 1.24% (w/v) sodium chloride and 0.56% (w/v) sodium carbonate (anhydrous).

This differential analysis was performed by addition of 50 microliters whole blood to a glass culture tube containing 300 microliters lyse reagent. The sample and lyse are mixed by swirling the contents of the tube for approximately 6 seconds and the lytic activity of the formic acid arrested by addition of 165 microliters of the above quench. The sample was then subjected to electro-optical measurement in the manner described in Example II and the resultant differential analysis was comparable to that of Example II, (as illustrated in FIGS. 1, 2 and 3).

EXAMPLE V

The procedures of Example I were repeated, except for the substitution of 0.1% (v/v) acetic acid for the formic acid in the lytic reagent system. The lytic activity of the reagent was arrested, after approximately 5 seconds, with a quench comprising 0.25% sodium bicarbonate in 2.0% sodium chloride solution. The sample was adequately quenched and ready for differential analysis by flow cytometry techniques within about 5 to 10 seconds subsequent to the addition of the quench. The equipment and analytical techniques used in such analysis were essentially the same as in Example I. The differential analysis performed on this sample is illustrated in the scattergram of FIG. 5.

EXAMPLE VI

The procedures of Example V were repeated except for extending the period of contact of the sample and the lytic reagent from 5 to 7 seconds prior to the addition of the quench. The quench was also modified slightly by increasing the concentration of sodium bicarbonate from 0.25% to 0.375% in the 2.0% sodium chloride solution. The results of this differential analysis of the leukocyte population are illustrated in the scattergram of FIG. 6.

EXAMPLE VII

The procedures of Example I are repeated, except for the separation and identification of the various sub-populations of leukocyte by immunochemical techniques. Once the action of the lytic reagent is quenched, the sample is diluted with an isotonic buffer and sequentially slurried with a series of magnetic particles, each of which having been pre-treated with a different antiserum specific for adsorption of only one species of leukocytes. The adsorbed cells can be separated sequentially from the sample using the conventional magnetic particle separation techniques described in the previously incorporated references. The separated particles can then undergo additional screening for surface markers which are indicative of one or more disease states.

EXAMPLE VIII

The procedures of Example VII are repeated, except that the cells are separated from the magnetic particles and cultured with an immortal cell line in accordance with the procedures of Kohler and Milstein, Nature, Vol. 256, 495-497 (1975). The clones produced in this fashion are screened for the production of antiserum specific for the surface marker of interest.

The foregoing Detailed Description and Examples are intended as illustrative of some of the preferred embodiments of the lytic reagents, reagent systems and methods of this invention. It is not the intent of the inventors that these specific embodiments of their invention be interpreted as indicative of the scope or breadth thereof, but rather simply supportive of the claims which are set forth hereinafter.

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US3446751 *Nov 29, 1965May 27, 1969Brunswick CorpHemolyzing composition
US3859098 *Jul 13, 1972Jan 7, 1975Konishiroku Photo IndPhotoresist composition
US3872166 *Jun 25, 1973Mar 18, 1975Toni DocknerImprovement in the recovery of glyoxylic acid
US3874852 *Jul 5, 1974Jul 11, 1989 Title not available
US3896107 *Mar 8, 1974Jul 22, 1975Hercules IncDerivatized ethyl cellulose
US3941729 *Apr 28, 1975Mar 2, 1976Normac, Inc.Styrene suspension polymerization system for spheres
US4099917 *Jul 21, 1977Jul 11, 1978Technicon Instruments CorporationProcess for preparing a cell suspension from blood for discrimination of white blood cells and platelets from other blood particles
US4213876 *Aug 22, 1978Jul 22, 1980Coulter Electronics, Inc.Multi-purpose blood diluent for use in electronic blood analysis instrumentation
US4250051 *Dec 26, 1978Feb 10, 1981Coulter Electronics, Inc.Preservative for use in calibrator compositions for blood analysis
US4286963 *Nov 23, 1979Sep 1, 1981Coulter Electronics, Inc.By lysis of erythrocytes using a quaternary amonium compound and an alcohol compound, and measurement of hemoglobin by chromogen formation
US4324685 *Mar 21, 1980Apr 13, 1982Beckman Instruments, Inc.Water soluble amine chloride
US4333873 *Apr 24, 1981Jun 8, 1982Eli Lilly And CompanyAnalgesics, tetrapeptides enkephalin analogs
US4346018 *Jun 16, 1980Aug 24, 1982Coulter Electronics, Inc.Stabilization of components
US4379708 *Feb 18, 1982Apr 12, 1983Rego Norberto O SProcess for tanning fish skins
US4436821 *Apr 12, 1982Mar 13, 1984Streck Laboratories, Inc.Simulated human platelets from red blood cells
US4448722 *Sep 27, 1982May 15, 1984The Hilton-Davis Chemical Co.Acid addition salts of copper phthalocyanine-used in dyeing cellulose
US4485175 *Jan 3, 1983Nov 27, 1984Coulter Electronics, Inc.Quaternary ammonium salts as lysing reagent
US4637986 *Aug 22, 1983Jan 20, 1987Ortho Diagnostic Systems, Inc.Flow cytometry lysing reagent with leukoprotective agent for producing a 3-part WBC count
US4751179 *May 31, 1984Jun 14, 1988Coulter Electronics, Inc.Method and reagents for differential determination of four populations of leukocytes in blood
US4902613 *Dec 8, 1986Feb 20, 1990Becton, Dickinson And CompanyLysing agent for analysis of peripheral blood cells
CA1087966A1 *Jun 25, 1976Oct 21, 1980Jacques BenvenisteProcess and a metachromatic composition for effecting a count of leucocytes, and more particularly of basophiles
JPS5468291A * Title not available
Non-Patent Citations
Reference
1DaCosta, A. J., Transfusion, vol. 13, No. 5, 1973 "Effects of Ethacrynic Acid on Human Red Blood Cells", pp. 305-313.
2 *DaCosta, A. J., Transfusion, vol. 13, No. 5, 1973 Effects of Ethacrynic Acid on Human Red Blood Cells , pp. 305 313.
3Miale, John B., "Laboratory medicine Hematology", Third Edition p. 1122, 1967, Published by C. V. Mosby Co.
4 *Miale, John B., Laboratory medicine Hematology , Third Edition p. 1122, 1967, Published by C. V. Mosby Co.
5 *Rother, U., Z. Immun. Forsch., vol. 155, 1978, Deviated Lysis: Lysis of Unsensitized Cells by Complment. V. Generation of the Activity by Low pH or Low Ionic Strength , pp. 118 129.
6Rother, U., Z. Immun.-Forsch., vol. 155, 1978, "Deviated Lysis: Lysis of Unsensitized Cells by Complment. V. Generation of the Activity by Low pH or Low Ionic Strength", pp. 118-129.
7Schettini, F., Acta Pardiat. Scand., vol. 60, 1971 "Acid Lysis of Red Blood Cells in Normal Children", pp. 17-21.
8 *Schettini, F., Acta Pardiat. Scand., vol. 60, 1971 Acid Lysis of Red Blood Cells in Normal Children , pp. 17 21.
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US5262329 *May 3, 1993Nov 16, 1993Carver Jr Edward LMethod for improved multiple species blood analysis
US5486477 *Aug 11, 1993Jan 23, 1996Carver, Jr.; Edward L.Reagent system for improved multiple species blood analysis
US5516695 *Aug 29, 1994May 14, 1996Abbott LaboratoriesMultipurpose reagent system for rapid lysis of whole blood
US5618733 *Dec 20, 1994Apr 8, 1997Toa Medical Electronics Co., Ltd.Ionic surfactant for lysing erythrocytes and causing damage to leukocyte cell membranes, organic compound having acidic group
US5648225 *Aug 25, 1994Jul 15, 1997Abbott LaboratoriesMethod of using a multi-purpose beagent for subclassification of nucleated blood cells
US5686308 *Jun 8, 1995Nov 11, 1997Coulter CorporationEthoxylated long chain amine salt
US5691204 *Apr 21, 1995Nov 25, 1997Abbott LaboratoriesCompositions and methods for the rapid analysis of reticulocytes
US5786224 *Jun 29, 1995Jul 28, 1998Coulter CorporationAnionic surfactants, nonionic surfactants, leukocytes in blood sampling, acidification with formic acid with other acids
US5817518 *Dec 18, 1995Oct 6, 1998Coulter International Corp.Reagent and method for differential determination of leukocytes in blood
US5843608 *Nov 4, 1997Dec 1, 1998Coulter International Corp.Reagent and method for differential determination of leukocytes in blood
US5874310 *Nov 21, 1997Feb 23, 1999Coulter International Corp.Method for differentiation of nucleated red blood cells
US5874311 *Nov 21, 1997Feb 23, 1999Coulter International Corp.Simple and efficient quantitative analysis of blood sample by cytolysis of mature erthyrocytes, measuring low angle light scatter signal without staining or fluorescence
US5882933 *Jul 25, 1997Mar 16, 1999Coulter International Corp.Method for determination of leukocytes and hemoglobin concentration in blood
US5917584 *Jan 22, 1998Jun 29, 1999Coulter International Corp.Method for differentiation of nucleated red blood cells
US6210969Apr 28, 1999Apr 3, 2001Coulter International Corp.Lytic reagents for automatically determining leukocyte subpopulations in blood, selectively lysing red blood cells
US6214625Apr 28, 1999Apr 10, 2001Coulter International Corp.Composition and method for differentiation of basophils and eosinophils in blood
US6246786 *Sep 17, 1997Jun 12, 2001Sysmex CorporationParticle analyzer
US6472215Jul 27, 2001Oct 29, 2002Coulter International Corp.Analyzes blood cell distribution patterns obtained from two separate blood measurement results, determines the presence of nucleated red blood cells, and further enumerates nucleated red blood cells in a blood sample
US6514763 *Mar 23, 2001Feb 4, 2003Hematronix, Inc.Leukocytes, lytic agent (e.g., alkanoic acid), quench, and post-lytic fixing agent (e.g., propylene glycol)
US6743634 *Aug 23, 2002Jun 1, 2004Coulter International Corp.Irradiate the cells and particles, one at a time, with a beam of radiation, and to detect back-scattered (reflected) radiation using a plurality of optical fibers to transmit the back-scattered radiation to a high-gain photodetector,
US6762055Jan 6, 2003Jul 13, 2004Bio-Rad Laboratories, Inc.Method for preparation of hematology blood control
US6869569Apr 21, 2004Mar 22, 2005Coulter International Corp.Especially platelets and basophils, on the basis of their back-scatter signature;
US7064823May 31, 2002Jun 20, 2006Idexx Laboratories, Inc.Consumable tube for use with a flow cytometry-based hematology system
US7247484Oct 31, 2003Jul 24, 2007Streck, Inc.Hematology reagent and methods
US7324194May 10, 2006Jan 29, 2008Idexx Laboratories, Inc.Flow-cytometry-based hematology system
US7413905 *Sep 7, 2006Aug 19, 2008Shenzhen Mindray Biomedical Electronics Co., Ltd.Reagents and methods for classifying leukocytes
US7416891 *Sep 7, 2006Aug 26, 2008Shenzhen Mindray Bio-Medical Electronics Co., LtdReagent and method for classifying leucocytes
US7449337Jan 24, 2007Nov 11, 2008Idexx Laboratories, Inc.Lytic reagent and method for leukocytes differential in whole blood
US7709653Jun 10, 2009May 4, 2010Shenzhen Mindray Bio-Medical Electronics Co. Ltd.asymmetric cyanine dye containing e.g.1-(2-hydroxyethyl)-4-methylquinolinium bromide; for staining biological samples selected from nucleic acids, erythroblasts or reticulocytes in the blood
US7867765 *Dec 28, 2006Jan 11, 2011The General Hospital CorporationBlood cell sorting methods and systems
US7960099Dec 31, 2007Jun 14, 2011Shenzhen Mindray Bio-Medical Electronics Co., Ltd.White blood cell differentiation reagent containing an asymmetric cyanine fluorescent dye and method of use thereof
US8067602Dec 12, 2008Nov 29, 2011Shenzhen Mindray Bio-Medical Electronics Co., Ltd.Asymmetric cyanine fluorescent dyes, compositions and their use in staining biological samples
US8143064Nov 5, 2008Mar 27, 2012Shenzhen Mindray Bio-Medical Electronics Co., Ltd.Reagents and methods for classifying leukocytes
US8163559Jan 28, 2011Apr 24, 2012Shenzhen Mindray Bio-Medical Electronics Co., Ltd.White blood cell differentiation reagent and method
US8187886Jan 10, 2011May 29, 2012The General Hospital CorporationBlood cell sorting methods and systems
US8273329Jun 29, 2010Sep 25, 2012Shenzhen Mindray Bio-Medical Electronics Co., Ltd.Cyanine compounds, compositions including these compounds and their use in cell analysis
US8304243Mar 23, 2012Nov 6, 2012Shenzhen Mindray Bio-Medical Electronics Co., Ltd.Reagents and methods for classifying leukocytes
US8334109Dec 8, 2009Dec 18, 2012Shenzhen Mindray Bio-Medical Electronics Co., Ltd.Reagent for blood analysis and method of using the same
US8367358Sep 28, 2009Feb 5, 2013Shenzhen Mindray Bio-Medical Electronics Co., Ltd.Reagent, kit and method for differentiating and counting leukocytes
US8383830Oct 28, 2009Feb 26, 2013Shenzhen Mindray Bio-Medical Electronics Co., LtdCyanine compounds and their use in staining biological samples
US8486629Apr 26, 2006Jul 16, 2013Bioarray Solutions, Ltd.Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation
US8486720Aug 3, 2004Jul 16, 2013Bioarray Solutions, Ltd.Arrays of magnetic particles
US8563247Jul 29, 2010Oct 22, 2013Bioarray Solutions, Ltd.Kits for multiplexed nucleic acid analysis by capture of single-stranded DNA produced from double-stranded target fragments
US8615367Mar 24, 2011Dec 24, 2013Bioarray Solutions, Ltd.Number coding for identification of subtypes of coded types of solid phase carriers
US8685661Jul 26, 2010Apr 1, 2014Shenzhen Mindray Bio-Medical Electronics Co., Ltd.Reagent and kit for classifying and counting leukocytes, the preparation thereof, and process for classifying and counting leukocytes
US8691594Oct 31, 2012Apr 8, 2014Bioarray Solutions, Ltd.Method of making a microbead array with attached biomolecules
US8691754Jun 7, 2010Apr 8, 2014Bioarray Solutions, Ltd.Microparticles with enhanced covalent binding capacity and their uses
US8712123Apr 12, 2011Apr 29, 2014Bioarray Solutions, Ltd.Analysis, secure access to, and transmission of array images
US8753888May 25, 2012Jun 17, 2014The General Hospital CorporationBlood cell sorting methods and systems
US20110275111 *Jan 26, 2010Nov 10, 2011Koninklijke Philips Electronics N.V.Microfluidic device for full blood count
USRE42143Mar 6, 2007Feb 15, 2011Idexx Laboratories, Inc.Consumable tube for use with a flow cytometry-based hematology system
EP0695936A2 *Jun 16, 1995Feb 7, 1996TOA MEDICAL ELECTRONICS CO., Ltd.A method for classifying and counting leukocytes
EP2259044A1Jun 5, 2009Dec 8, 2010Koninklijke Philips Electronics N.V.Multi-frequency impedance method and apparatus for discriminating and counting particles expressing a specific marker
EP2259045A1Jun 5, 2009Dec 8, 2010Koninklijke Philips Electronics N.V.Multi-frequency impedance method and apparatus for discriminating and counting particles expressing a specific marker
WO1996034283A1 *Apr 24, 1996Oct 31, 1996Hematronix IncLytic system utilizing propionic acid for leukocytes differentiation
WO1996042015A2 *May 24, 1996Dec 27, 1996Coulter Int CorpReagent and method for differential determination of leukocytes in blood
WO2010140127A1Jun 2, 2010Dec 9, 2010Koninklijke Philips Electronics N.V.Multi -frequency impedance method and apparatus for discriminating and counting particles expressing a specific marker
Classifications
U.S. Classification436/17, 436/63, 436/10, 436/18
International ClassificationG01N33/50, G01N33/569
Cooperative ClassificationG01N33/5094, G01N33/56972
European ClassificationG01N33/50D6, G01N33/569H2
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